Lipoprotein(a) (Lp(a))[1]

Lipoprotein(a) (Lp(a)), an independent, causal cardiovascular risk factor, is a lipoprotein particle that is formed by the interaction of a low-density lipoprotein (LDL) particle and apolipoprotein(a) (apo(a))1,2. Apo(a) first binds to lysine residues of apolipoprotein B-100 (apoB-100) on LDL through the Kringle IV (KIV) 7 and 8 domains, before a disulfide bond forms between apo(a) and apoB-100 to create Lp(a) (refs. 3-7). Here we show that the first step of Lp(a) formation can be inhibited through small-molecule interactions with apo(a) KIV7-8. We identify compounds that bind to apo(a) KIV7-8, and, through chemical optimization and further application of multivalency, we create compounds with subnanomolar potency that inhibit the formation of Lp(a) [2].

Oral doses of prototype compounds and a potent, multivalent disruptor, LY3473329 (muvalaplin), reduced the levels of Lp(a) in transgenic mice and in cynomolgus monkeys. Although multivalent molecules bind to the Kringle domains of rat plasminogen and reduce plasmin activity, species-selective differences in plasminogen sequences suggest that inhibitor molecules will reduce the levels of Lp(a), but not those of plasminogen, in humans. These data support the clinical development of LY3473329—which is already in phase 2 studies—as a potent and specific orally administered agent for reducing the levels of Lp(a) [2].

LPA transgenic mouse studies[2]
The LPA gene (GenBank accession code NM_005577) encoding the human apo(a) protein with a signal peptide, one KIV type 1, six KIV type 2 repeats, one each of KIV types 3–10, one KV and one protease domain was subcloned into a transgenic vector containing a mouse albumin promoter cassette and a human growth hormone polyadenylation signal. The transgenic mouse line was generated by pronucleus injection of the LPA transgenic vector. Eight individual transgenic founder mice were assessed for germline transmission of LPA by genotyping and assessment of the levels of apo(a) in the plasma. Positive founder mice were crossed with ApoB100 transgenic mice (Taconic model 1004), and the resulting mouse line nomenclature (B6.SJL-Tg(APOB)1102Sgy Tg(Alb-LPA)32Arte) was selected on the basis of the confirmation of germline transmission, the identification of LPA and huApoB100 transgenes and the levels of Lp(a) in the plasma. Mice hemizygous for both transgenes were used for pharmacology studies. The studies were conducted in female Lp(a) double transgenic mice (age around 7–17 months). Mice were housed in cages with a standard light cycle (12-h light, 12-h dark), at room temperature (22 ± 4 °C), with a relative humidity of 30–70%. Mice were identified by numbers on the cage cards. After arrival, mice were fed on a normal diet (Harlan Teklad diet 2014). Mice were randomized to treatment groups (n = 5 per group, unless otherwise noted) by body weight and baseline plasma Lp(a) concentration using a block randomized allocation tool (BRAT) for the study. In an example Lp(a) transgenic mouse study in which 66 mice were prescreened for baseline Lp(a) levels, the average Lp(a) level was 45 ± 1.8 µg ml−1 (range 20–78 µg ml−1). Mice were dosed with various compounds in vehicle (10 ml kg−1, 1% hydroxyethyl cellulose (HEC), 0.25% Tween-80, Antifoam) or with vehicle alone as a control orally BID for five days. Blood samples were collected by tail bleeds at designated times, and Lp(a) levels were determined by ELISA by an investigator who was blinded to group allocation. In brief, lipoprotein particles were captured by a goat anti-Lp(a)-antibody-coated plate (Abcam ab31675, diluted 1:12,500 in HEPES-buffered saline), the plates were washed and samples were detected using an HRP-conjugated goat anti-apoB antibody (Abcam ab27622, diluted 1:3,000). Colorimetric peroxidase substrate 3,3’,5,5’-TMB was added, and the reaction was stopped using 1 N sulfuric acid. Absorbance at 450 nm was read on a Molecular Devices SpectraMax plate reader. Blood samples were collected at four hours after dosing on day 5 of dosing to confirm exposure. Full pharmacokinetic (PK) profiles were not evaluated. Concentrations of compounds were evaluated from dried blood spots through a non-Good Laboratory Practice (GLP) liquid chromatography with tandem mass spectrometry (LC–MS/MS) assay. Data were analysed in GraphPad Prism v.9.5.1.[2]
Cynomolgus monkey studies[2]
Female or male Chinese Macaca fascicularis cynomolgus monkeys (as described in the figure legends) of unspecified ages with body weight ranging from 2.0 kg to 5.0 kg were housed in cages with a standard light cycle (12-h light, 12-h dark) at room temperature (20–26 °C); the relative humidity ranged from 30% to 70%. Monkeys were given fruits, vegetables or dietary enrichment as a form of environmental enrichment, and various cage enrichment devices. Purina Lab diet 5048C was provided BID and individual monkeys were identified by cage card. The monkeys were randomized to treatment groups by body weight and baseline plasma Lp(a) concentration, as determined by a commercially available Randox assay (Randox LP2757), using a BRAT for the study. In an example cynomolgus monkey study in which 39 monkeys were prescreened for baseline Lp(a) levels, the average Lp(a) level was 350 ± 57 µg ml−1 (range 53–1,667 µg ml−1). Monkeys were dosed orally with vehicle (purified water) or compounds QD or BID for 14 days, at dose levels and frequencies as previously described for each compound. Plasma samples were collected on days before dosing to establish baseline Lp(a) and after the morning dose during the study. Lp(a) levels were measured by an investigator who was blinded to group allocation using the Randox assay, and were quantified relative to the calibrator series (Randox LP3404). Data were analysed in GraphPad Prism v.9.5.1. The Lp(a) percentage change from before dosing in non-human primates was analysed by repeated measures ANOVA in SAS v.9.4 (SAS). At each time point, the Lp(a) percentage change for each treated group was compared with vehicle by the Bonferroni method. Plasma samples were collected from all monkeys before dosing and at four-hour time points only on days 1, 3, 5, 9 and 14. A full PK profile was obtained in the first three monkeys of each dose group on day 15, collecting samples before dosing and 1, 2, 4, 8, 12, 24, 48 and 96 h after dosing. Concentrations of compounds were evaluated in plasma samples using a non-GLP LC–MS/MS assay. The non-compartmental plasma PK parameters were calculated using Watson (v.7.5).

[1]. Bhatia HS, et al. Lipoprotein(a): Evidence for Role as a Causal Risk Factor in Cardiovascular Disease and Emerging Therapies. J Clin Med. 2022 Oct 13;11(20):6040.
[2]. Discovery of potent small-molecule inhibitors of lipoprotein(a) formation. Nature. 2024 May 8. doi: 10.1038/s41586-024-07387-z.

C42H54N4O6

710.901371479034

710.4

C, 70.96; H, 7.66; N, 7.88; O, 13.50

2565656-70-2

155369486

Off-white to light yellow solid

-2.8

6

10

18

52

1020

6

C1CNC[C@H]1[C@H](CC2=CC(=CC=C2)CN(CC3=CC=CC(=C3)C[C@@H]([C@H]4CCNC4)C(=O)O)CC5=CC=CC(=C5)C[C@@H]([C@H]6CCNC6)C(=O)O)C(=O)O

BRLGERLDHZRETI-BGBFCPIGSA-N

InChI=1S/C42H54N4O6/c47-40(48)37(34-10-13-43-22-34)19-28-4-1-7-31(16-28)25-46(26-32-8-2-5-29(17-32)20-38(41(49)50)35-11-14-44-23-35)27-33-9-3-6-30(18-33)21-39(42(51)52)36-12-15-45-24-36/h1-9,16-18,34-39,43-45H,10-15,19-27H2,(H,47,48)(H,49,50)(H,51,52)/t34-,35-,36-,37-,38-,39-/m0/s1

(2S,2'S,2''S)-3,3',3''-((nitrilotris(methylene))tris(benzene-3,1-diyl))tris(2-((R)-pyrrolidin-3-yl)propanoic acid)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 11.36 mg/mL (15.98 mM)
H2O: < 0.1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)